人脐带间充质干细胞移植治疗角膜碱烧伤的实验研究

Experimental study of transplantation of human umbilical cord mesenchymal stem cells in the treatment of alkali-burned cornea

摘要:

目的：研究人脐带间充质干细胞(hUCMSCs)移植治疗兔角膜碱烧伤的疗效，分析多形核中性白细胞(PMNs)的浸润和血管内皮生长因子(VEGF)的表达变化。

方法：将75只健康日本白兔随机分成A、B、C组，每组25只，均建立右眼角膜碱烧伤模型。A组兔右眼角膜碱烧伤后立即行角膜病灶区羊膜负载hUCMSCs覆盖术； B组兔右眼角膜碱烧伤后立即行角膜病灶区羊膜覆盖术； C组兔右眼角膜碱烧伤后未进行处理。角膜碱烧伤后3、7、14、21、28d，裂隙灯下观察实验兔角膜恢复情况并照相，对角膜新生血管(CNV)生长情况进行评分，并分离角膜组织制作病理切片，通过苏木素-伊红(HE)染色观察PMNs浸润情况，采用免疫组化染色法测定VEGF的表达。

结果：角膜碱烧伤后14d时，A组CNV较B组生长明显缓慢，A组病灶区周围CNV生长评分明显低于B组(P<0.05)。碱烧伤后3d时，角膜PMNs数量增加，浸润角膜基质层，7d时稍有下降，14d时达到峰值，后渐进性下降，碱烧伤后早期浸润在病灶区角膜基质内，后期浸润范围与溃疡面积相同，碱烧伤后各时间点A组和B组角膜PMNs密度均明显低于C组(P<0.05)，且14、21d时，A组均明显低于B组(P<0.05)。碱烧伤后各组角膜VEGF表达水平均在7～14d时达到峰值，28d时明显降低，碱烧伤后各时间点A组和B组VEGF表达水平均明显低于C组(P<0.05)，且7、14、21d时，A组均明显低于B组(P<0.05)。

结论：羊膜负载hUCMSCs移植治疗兔角膜碱烧伤可减少CNV形成，抑制碱烧伤后角膜血管化，角膜病理损伤及血管化与PMNs和VEGF密切相关。

Abstract:

AIM: To evaluate the efficacy of transplantation of human umbilical cord mesenchymal stem cells(hUCMSCs)in the treatment of corneal alkali burn in rabbits, and study the infiltration of polymorphonuclear neutrophils(PMNs)and the changes of vascular endothelial growth factor(VEGF)expression.

METHODS: Corneal alkali burn models were established in right eyes of 75 healthy Japanese white rabbits, which were divided into three groups(group A, B and C), with 25 rabbits in each group. Group A was treated with amniotic membrane combined with hUCMSCs on the day after corneal alkali burn. Group B was treated with amniotic membrane only. Group C did not give any treatment after corneal alkali burn. At 3, 7, 14, 21 and 28d after corneal alkali burn, the corneal recovery was observed by slit lamp and photographed, the growth of corneal neovascularization(CNV)was scored, and corneal tissue was separated to make pathological sections. PMNs infiltration was observed by hematoxylin-eosin(HE)staining, and the expression of VEGF was determined by immunohistochemical staining.

RESULTS: The growth of CNV in group A was much slower than that in group B at 14d after alkali burn. The CNV growth score around lesions of group A was significantly lower than that of group B(P<0.05). The quantity of PMNs increased on the 3d with the stromal layer of cornea infiltrated, relatively decreased on the 7d, shown a peak on the 14d, and then decreased gradually. Early infiltration after alkali burn was in the corneal stroma of the lesion area, and the extent of infiltration was equal to the ulcer area at later stage. The cell densities of corneal PMNs in group A and group B were significantly lower than those in group C at all time points after alkali burns(P<0.05), and those in group A were significantly lower than group B at 14 and 21d(P<0.05). The expression levels of corneal VEGF in all groups after alkali burn reached peak at 7～14d and decreased significantly at 28d, and the expression levels of VEGF in group A and group B at all time points after alkali burn were significantly lower than those in group C(P<0.05), and group A was significantly lower than that in group B at 7, 14 and 21d(P<0.05).

CONCLUSION: The transplantation of hUCMSCs after alkali burn cornea can reduce the formation of CNV and inhibit corneal revascularization after alkali burn. The corneal pathological lesions and vascularization are closely related to PMNs and VEGF.